Group A rotavirus infections are the most common cause of severe gastroenteritis among infants and young children worldwide. The rhesus rotavirus RRV-based quadrivalent vaccine developed in this laboratory was licensed by FDA in 1998. A second generation vaccine, the bovine rotavirus UK-based quadrivalent vaccine has been undergoing phase I-II human trials. Rotavirus surface proteins VP4 and VP7 have been shown to be independent protective antigens. Recently, in studies by others in a mouse model, (i) certain IgA monoclonal antibodies directed to rotavirus inner capsid protein VP6 were reported to be protective against disease, and (ii) antibodies directed to rotavirus nonstructural protein NSP4 (viral entertoxin) were shown to be effective passively in protection against disease. More recently, an immunohistochemistry assay was developed, in which Sf-9 cells infected with recombinant baculovirus that expresses a specific rotavirus protein as an antigen was utilized to assess immune response. In order to establish an immunohistochemistry assay in our laboratory for assessment of immune response to selected rotavirus proteins following natural rotavirus infection or vaccination, we constructed recombinant baculoviruses expressing rotavirus (i) outer capsid protein VP7 of human rotavirus (HRV) D (G1), DS-1 (G2), P (G3) and ST3 (G4), (ii) outer capsid VP4 of RRV and bovine UK, and (iii) nonstructural protein NSP4 of HRV Wa and DS-1, RRV and porcine SB-1A. Sera from gnotobiotic piglets or calves infected orally with selected human or animal rotaviruses were analyzed by immunocytochemistry assay for homotypic and heterotypic serum IgG responses to various recombinant-expressed VP4 proteins including VP4 (P) serotype 1A, 1B, 2A, 3, 4, 5B and 7. In primary infections, antibody responses to homologous VP4 were significantly higher than those to heterologous VP4s. Heterotypic antibody levels to animal rotavirus VP4s (P5B[RRV] or P7[UK]) in animals infected with HRVs (P1A or P1B) were low. Similarly, heterotypic responses to HRV VP4s (P1A, 1B, 2A, 3 or 4) induced by RRV VP4 (P5B) or UK VP4 (P7) were of low titer or infrequent. Piglets infected with one HRV (P1A) in utero and challenged postnatally with another HRV (P1A) developed significant and high mean antibody responses to both homologous and heterologous VP4s except for P3 VP4. A significant and relatively high homotypic (P1A) mean antibody response was observed in piglets infected with RRV (P5B) at birth and challenged 2 weeks later with HRV (P1A). No significant mean antibody responses to P1B, P2A, P3, P4, P5B or P7 VP4 were observed in this group. - Rotavirus, infantile diarrhea, baculovirus recombinant, immunocytochemistry assay